Cloning multiple control sequences into chromosomes or into artificial centromeres

ABSTRACT

Artificially synthesizing 29 homozygous cystic fibrosis core panel controls demonstrates placing multiple homozygous mutant sequences on the same single control DNA sequence to streamline quality control by minimizing extra control assays, time, and costly formatted test materials and testing all controls during every test. Any rare or unavailable reported DNA sequence can be PCR amplified using primer pairs synthesized with the designated mutation or variant sequence with paired adjacent upstream and downstream primers to amplify target sequences in total genomic DNA.

SUMMARY

When tested together, twenty nine artificially synthesized, cloned,homozygous cystic fibrosis controls streamline test quality control byminimizing control assay number, cost, and assay time. Any rare orunavailable reported sequence can be PCR amplified from total genomicDNA or RNA template using a synthesized primer incorporating the mutant,polymorphic or variant sequence and a paired upstream or downstreamprimer. These synthesized upstream and downstream fragments overlap atthe modified site and are amplified together to obtain a homozygousmutant control. The 5′ and 3′ flanking sequences were selected to spanall the PCR sites in the test platforms we were using. In this manner 1to 4 homozygous mutations were artificially synthesized into each of 17fragments 433 bp-933 bp long by PCR amplification on total normal humangenomic DNA template. Then one to three synthesized fragments were bluntend cloned into 9 vectors and validated by sequencing. Together thesemutations included (1) 7 mutations on two joined fragments cloned into asingle vector's multiple cloning site and (2) all 25 homozygousmutations originally recommended by ACMG for the core cystic fibrosispanel which are sufficiently long to be amplified by the PCR primerpairs in most commercial platforms. Together these cloned mutantsequences provide controls for each PCR assay to optimize multiplex testreliability for all recommended ACMG mutations.

Key words: controls: homozygous, synthetic, PCR amplified, multiplex

Achieving a reliability of 99% for any 25 mutation test requires anindividual mutation test reliability of 99.96% (1 incorrect result among2500 reported results). In contrast, achieving a test reliability of 99%for a 100 mutation test requires an individual mutation test reliabilityof 99.99% (1 incorrect among 10,000 reported results). When applyingever larger multiplex tests, simultaneously testing controls for alltarget sequences is required to maintain the most reliable moleculargenetic analyses. Thus the College of American Pathologists' MolecularGenetics Committee dictates that whenever possible a control needs to beincluded in an independent control reaction for each molecular assay andthat the best available control is to be used. At the same time,obtaining controls for each reported gene and its common mutations istypically the most difficult hurdle required to introduce any new assayinto a Clinical Molecular Genetics laboratory's test menu. The followingprotocol demonstrates the relative ease required to synthesize controlsequences of standard PCR amplifiable lengths for use by multiplelaboratory specific and commercial multiplex cystic fibrosis testplatforms.

The cystic fibrosis transmembrane receptor (CFTR) gene is mutated inboth alleles in patients with cystic fibrosis (CF [MIM 21977]) andcongenital bilateral aplasia of vas deferens (CBAVD [MIM 277180]).Cystic fibrosis is the most common lethal genetic disease in Caucasians,affecting about 1 in 2500 newborns while CBAVD renders about 1 in 5,000males sterile with about half of these patients exhibitingcystic-fibrosis like symptoms and a few have full-blown cystic fibrosis.Over 1000 mutations and 50 polymorphisms have been reported worldwidethroughout a large portion of this 24 exon CFTR gene with its 4600basepair coding sequence that spans 188 kb of genomic DNA (CysticFibrosis Mutation Database.)

The ACMG 25 mutation cystic fibrosis panel was selected by the CysticFibrosis Committee of the American College of Medical Genetics forscreening pregnant Caucasian women who are at a more substantial risk ofhaving a cystic fibrosis fetus than women from other races (Grody et al.2001). These selected 25 CFTR mutations included all mutations with afrequency of at least 0.1% in cystic fibrosis patients. This wasconsidered an attainable goal for a large number of laboratories tooffer routinely and at the same time charge a reasonable fee toencourage third party payment for their laboratory service. Currentlythousands of pregnant Caucasian patients are tested each month by dozensof laboratories as a first step in detecting most fetuses at-risk forcystic fibrosis.

Although initially developed for population carrier screening, the 25mutation core panel can also be used to screen patients with suspicioussymptoms because over 90% of all affected Caucasian patients will testpositive for at least one mutation including over 99% of affectedNorthern European Caucasian patients (Lebo and Grody, unpublished data).We incorporated the 5T allele into the select group of 25 most commonmutations tested in symptomatic patients because (1) the 5T allele withthe severe ΔF508 mutation and the 5T allele with decreased penetranceare the two most common alleles resulting in cystic fibrosis-likesymptoms including CBAVD, (2) 0.17% to 3.4% of cystic fibrosis patientswith severe symptoms have one allele with the 5T sequence without anyother detected mutation, and (3) about 40% of CBAVD patients arecompound heterozygotes for 5T and another CFTR gene mutation (Claustreset al. 2000; Kerem et al. 1997; Lebo and Grody, unpublished data).

When the 25 mutation panel was selected by the ACMG Cystic FibrosisCommittee, the controls for each of these mutations were unavailable inany one location. Thus many laboratories expanded their CFTR mutationtesting panel to 25 mutations and began offering the service withoutsimultaneously testing all the appropriate controls. In order to meetthe substantial new demand for better controls, Coriell Cell Repository,Camden, N.J., aggressively collected, transformed, and distributed humancell lines that contained all available mutations among those to betested. In spite of substantial efforts to date, Coriell has yet tomarket a collection of cell lines that together contain all 25mutations. Coriell has been marketing total genomic DNA from cell linestransformed with Epstein Barr virus with most of these represented asheterozygous mutations as a Product of Substantial Equivalence. Genzyme,which began testing about 87 mutations a decade ago, had to completemany thousands of tests over several years to identify and thenincorporate a patient DNA control for each of the 87 selected mutations.Furthermore, testing all 25 cystic fibrosis mutations in the Coriellcollection with each unknown set of patient samples requires asubstantial investment in labor and test materials. For this reasondozens of laboratories doing 25 mutation tests have been rotatingselected control patient DNAs from Coriell through their regularclinical protocol. In this fashion, all available mutation controls areonly tested among several independent multiplex assays run on differentdays, but all mutant sequences are never tested at the same time in asingle multiplex assay. Furthermore, most of the Coriell Cell lines areheterozygous at the CFTR mutation site so that these controls cannot bemixed together because the proportion of normal alleles increases eachtime another heterozygous genome is introduced. Therefore theseheterozygous controls must be used individually and cannot be used todetermine whether an assay distinguishes between a homozygous andheterozygous DNA sample, the primary deficiency in our studies (SeeDiscussion).

No matter how rare or unavailable, all selected reported mutations,polymorphisms, and variants can be synthesized not only as homozygousbut also as heterozygous controls using PCR primer pairs with mutantsequences, genomic DNA template, and editing DNA polymerase. Prior tooffering a 25 cystic fibrosis mutation test at Akron Children's Hospitalthree years ago, our laboratory artificially synthesized and verifiedhomozygous controls for all reported mutations by independent assays onthe Innogenetics multiplex format (FIG. 5). This strategy reduced theenergy and materials required to assay greater than a dozen controlreactions with permanent Coriell cell line DNAs to three InnogeneticsPCR amplification reactions and two ASR detection strips (Lebo et al.2003). Alternatively, mixing all homozygous mutations together resultedin a single multiplex control for the TM Biosciences platform (FIG. 6).Validation of these controls on other platforms was addressed by anotherstudy completed by Acrometrix Corp. (Benicia, Calif.; Aytay et al.,2005).

MATERIAL AND METHODS

Primers were selected incorporating published mutations into themutation-specific primers and selecting the background sequencesincluding the flanking primers from the CFTR gene sequences in TheGenome Database. All primers were synthesized by Invitrogen. Pfu DNAPolymerase or Pfu Ultra High-Fidelity DNA Polymerase was used for highfidelity PCR amplification (Stratagene, La Jolla, Calif.). All PCRamplifications were performed according to recommended assay conditionsand protocols for the selected enzyme (FIG. 1-III,V,VII,IX; See Results,for Synthetic Strategy). The PCR amplified products were sized byfluorescent ethidium bromide observation following agarose gelelectrophoresis. Many PCR reactions produced a single fragment of theexpected length, but when other fragments were also amplified, theappropriate fragment was purified by preparative agarose gelelectrophoresis prior to subsequent PCR amplification or cloning. Thepurified amplified fragments were quantified and blunt end cloned intocompetent bacteria. After growth and purification, the plasmid DNAdigested with appropriate restriction enzyme(s) was electrophoresed toconfirm that the insert had remained the correct length. The clones wereindependently validated by testing with one or two commercial cysticfibrosis test kits (Innogenetics, TM Biosciences; FIGS. 5,6) and bybidirectionally sequencing the cloned insert and its flanking vectorsequences (the Gold Standard for Clinical Molecular Genetics

Results

PCR Amplification with Synthesized Mutant Primers

Our goal was to design and synthesize a complete set of ACMG-recommendedhomozygous CFTR gene mutation controls for use in the widest variety ofcommercial and laboratory-specific CFTR platforms. Thus pairs of PCRprimer sites were selected to span all known primer sites reported toamplify each CFTR mutation-containing region (The Genome Database; FIG.1-I, F1 and R2). Then forward and reverse primers were selected to spanand incorporate one or more mutations between the flanking primer sites(FIG. 1-I, primers F2 and R1 with M1 and M2 mutation sites). The primerswere selected, synthesized, and used for PCR amplification of normalgenomic DNA template with primer pair F1 and R1 synthesizing themutation-containing upstream Product #1 (FIG. 1-II) and primer pair F2and R2 synthesizing the mutation-containing downstream Product #2 (FIG.1-III). Then the flanking forward and reverse primers F1 and R2 (FIG.1-III) were added to aliquots of Products #1 and #2 which were splicedtogether in a unique orientation by PCR amplification to give Product #3flanked by F1 and R2 sequences with the M1 and M2 mutations in themiddle (FIG. I-IV).

Additional mutations were added to a synthesized mutation-carryingfragment when another homozygous mutation was desired on the same genefragment. For example, when mutation M3 was desired on the same fragmentas mutations M1/M2, it was synthesized onto Product #3 template usingforward F3 and reverse R3 primers synthesized to include mutant sequenceM3 (FIG. 1-V to 1-VII). Analogous to the protocol to add the M1/M2mutations above, upstream Product #4 and downstream Product #5 weresynthesized using Product #3 as template prior to splicing thesetogether into Product #6 using flanking primers F1 and R2. Product #6now contains mutations M3 and M1/M2. The same protocol can be used toincorporate another homozygous mutation or substitute a new mutation foran existing mutation on Product #6 (FIG. 1,V-VIII).

Initially 31 of the first 34 manually selected primer pairs amplifiedthe correct target sequences. Three flanking primer sites that initiallyfailed to amplify a correct length unique product were moved to an evenmore distant location from the mutation site(s), thus assuring that thesynthesized mutation-carrying CFTR fragment would also span all theknown primer sites reported to amplify the gene region to be tested. All3 additional selected primers amplified the site for which each wasdesigned.

Our design included a plan to synthesize a small number of fragmentscontaining all 29 selected CFTR mutations that would provide an optimalmultiplex control according to the selected commercial or private formatusing the minimal number of control mixtures. The number of mutationsthat can be added to a single sequence is determined by the minimaldistance between mutations required to distinguish unambiguously betweennormal and mutant sequences tested by the selected assay formatincluding (1) nitrocellulose filters with slot blot designatedlocations, fluorescent beads, or microchips each with locations to whichASOs are hybridized uniquely, or (2) Mass Spec that hybridizescomplementary nucleotide sequences adjacent to the mutations to bemeasured and then adds additional 3′ nucleotides until a basecomplementary to the single base subtracted from the reaction mixture isencountered. For instance, all our homozygous controls are tested witheach assay to assure that each control unambiguously gives a homozygousmutant signal with no cross hybridization to the normal sequence.

When heterozygous controls are desired for applications like Mass Spec,multiple approaches can be used. For instance, normal genomic sequencescan be PCR amplified directly from total normal genomic template DNAusing the flanking primers like F1 and R2 (FIG. 1,I-IV). These amplifiednormal sequences can then be checked for size by electrophoresis,cloned, and each sequence verified by bidirectional sequence analysisthrough and beyond the vector's cloning site. Then these normal clonednormal gene sequences can be added to the cloned homozygous syntheticcontrols in equal copy number. Alternatively, Products #3A and #4A canbe PCR amplified together typically in equal concentrations usingforward primer F3 for the upstream mutation M3, reverse primer R1 forthe M1/M2 mutations, and F1 and R2 for the flanking primers (FIG.1,XI-XIV; Lebo et al., 2000).

The number of homozygous mutations that can be added to any single DNAfragment that can be tested unambiguously depends upon the assayapproach used by the selected CFTR test platform as well as theproximity of each mutations pair, the type and size of mutation(nucleotide substitution(s), insertion, or deletion), and theimmediately flanking sequences of each mutation. For instance, theIntron 10/Exon 11 fragment spans 5 common mutation sites: 1717-1G->A,G542X, G551D, R553X, and R560T (FIG. 2) while the ΔF507 and ΔF508mutations in Exon 10 overlap by 1 basepair and each delete threebasepairs. Thus the F507 and ΔF508 mutations which overlap by 1 basepairwere synthesized and cloned independently. Because the G551D and R553Xmutations are within 4 basepairs, these mutations were also synthesizedon independently cloned Intron 10/Exon 11 fragments that each carriedthree other mutations: 1717-1G->A, G542X and R560T. This design provideda means to mix these mutant sequences differently to optimize controlamplification and detection reaction number for both the commercialInnogenetics and TmBiosciences CFTR multiplex platforms (See below).

Following these strategies, 17 mutation-carrying fragments weresynthesized and tested with the Innogenetics assay to confirm that thesefragments carried the expected mutations. Some of the PCR fragmentprimers were designed to overlap with more than one independent fragmentto allow pairs of amplicons to be spliced together in the orientation ofchoice by PCR. Then the 17 fragments were spliced together into 9fragments and blunt end cloned. For instance, Product #9 (FIG. 1,X) wassynthesized using the same approach as Product #3 (FIG. 1,I-IV) exceptthat the upstream primer R2′F4 was selected in place of upstream primerF4. Then synthesized Product #9 is spliced to Product #6 by mixing andamplifying aliquots of these products together using flanking primers F1and R5 to synthesize Product #10 with mutations M1, M2, M3, and M4 (FIG.1,IX-X). Additional mutation-containing CFTR segments can be spliced toProduct #10 in this manner. Thus one PCR product was synthesized from 3joined fragments by repeating the splicing reaction to add a thirdmutation-carrying amplicon. In this fashion, the first 29 PCR amplifiedmutations on 17 synthesized fragments some of which were splicedtogether to form 9 fragments (FIG. 2).

Following the final PCR amplification and purification when required,the 9 spliced fragments were inserted into the cloning site of a singlevector. Inserts isolated from individual clones were screened for thecorrect size after restriction enzyme digestion. Then the presence ofthe synthesized CFTR mutation(s) and flanking sequences were detectedusing the Innogenetics CFTR multiplex mutation assay and furtheranalyzed by bidirectional sequencing of mini-prepped plasmid DNA. A 30%aliquot of a single miniprepped sample isolated from a 3 ml suspensionculture provided substantially more than sufficient material forbidirectional sequencing, validating two commercial platforms, andproviding complete controls for each of the thousands of cystic fibrosissamples tested at Akron Children's Hospital (Lebo et al. 2003).Bidirectional sequencing was completed by Cleveland Genomics (Cleveland,Ohio) using M13 sequencing primers hybridizing to these sites in thecloning vector and selected internal PCR primers used in the originalPCR synthesis. Each of the 9 fragments carried the expected subset of 29different cystic fibrosis mutations (FIG. 3). One of the nine clones hasan unreported 622-194G->A variation in intron 4 that is 284 basepairsremoved from the 711+1G->T mutation in exon 5. Three other variationsfound among these 6596 cloned DNA basepairs are found in the CFTR(cystic fibrosis transmembrane receptor) database (FIG. 3).

Three Insert Clone with A455E and N1303K Homologous Regions

In 15 of 17 synthesized, cloned, and sequenced fragments amplified fromtotal genomic DNA using synthesized DNA fragments to introduce 31 othermutant sites had only the primary CFTR sequence listed for the normalgene [CFTR Mutation Database] with the exception of three normallyvariant single nucleotide substitutions: 622-61A->T; 1525-61A->G, and405+46G->T. The first clone with three inserts had to be reengineered toreplace the first and third segments (FIG. 1, Bottom). The threesegments were: (Left) The 5′CFTR genomic segment with A455E from exon 9and 5T from intron 8; (Center) a segment with the 3849+10kbC->T mutationfrom intron 19; and (Right) the 3′ insert with the N1303K mutation fromexon 21. All of the sequences in the center segment except the inserted3849+10kbC->T site were identical to the corresponding normal CFTRgenomic sequence in the CFTR mutation database. In addition, 98% of theinitially amplified and cloned sequences in the 5′ A455E and 3′ N1303Kcontaining cloned segments were also identical to the correspondingnormal CFTR gene sequences in this database. However, 7 nucleotidesubstitutions downstream of the A455E site had been amplified from totalgenomic DNA that mimicked homologous sequences from the chromosome 20pseudogene and 11 nucleotides had been substituted from a 162 bpduplication downstream of the N1303K site in intron 21 (FIG. 5). Thusadditional clones from both the 3′ downstream segments from the A455Esite and from the N1303K site were reselected from the transformationplate, resequenced to find one with the correct genomic sequence, andsynthesized into the final clone that is being redistributed as part ofthe multiplex CFTR control sequences.

Validation on Two Commercial Platforms:

The Innogenetics multiplex CFTR mutation format coamplified threecombinations of these 9 clones each with unique CFTR mutationcombinations, and analyzed the amplified fragments on two sets ofmutation-specific ASOs bound to nitrocellulose filter paper. As part ofthis format validation, two different Intron10/Exon11 fragments weresequenced and tested: the first with G551D along with 1717-1G->A, G542X,and R560T, and the second with R553X along with 1717-1G->A, G542X, andR560T. When tested individually, the first fragment hybridized uniquelyto the G551D amplified control site as well as to the other threemutations (1717-1G->A, G542X, and R560T), but not to the normal siteR553 because the G551D mutation interferes with the binding to thenormal R553 sequence on the nitrocellulose filter strip (FIG.4,f1,left). In contrast, the second fragment with the R553X mutationbinds to both the R553X mutation site and also to the normal G551 site(FIG. 4,f3,right) because the R553X sequence does not preventhybridization to the G551 oligonucleotide at the hybridizationstringency and ASOs used in the assay. As illustrated, one otherhomozygous mutation was tested simultaneously with each of thesefragments (FIG. 4,f1,left and FIG. 4,f3,right) during the development ofthese specific cloned sequences. We also note that the 1717-1G->A mutantsite has a more intense signal than the other mutant sites G542X R553X,and R560T on the same fragment of DNA (FIG. 4,f3,right). We attributethis to different sensitivities of the assay for the PCR amplifiedsites.

Then all the fragments that were cloned and verified by bidirectionalsequencing (FIG. 2) were mixed appropriately, amplified in threeInnogenetics multiplex PCR reactions, and tested on two Innogeneticstest strips. These results detected homozygous mutant signal for all themutations tested (FIG. 4,mix,left and right). At the same time normalsignals were observed for the normal 394ΔTT, and 2143ΔT sites where thenormal CFTR gene sequence remained as well as the G551 site whichhybridized to the normal G551 labeled ASO in spite of the adjacent R553Xsequence modification. In contrast, the ΔF507 and ΔF508 mutationcontaining fragments were synthesized independently because thesefragments could not be introduced into the same normal CFTR genesequence at the same time. As expected, the Innogenetics ASOs analyzedboth the ΔF507 and ΔF508 mutations correctly without hybridizing to theF507 and F508 normal sequence (FIG. 4,mix,right). Therefore each of thetwo Innogenetics test strips gave exactly the results anticipated giventhe sequence characteristics and the prior results obtained when theclones were analyzed individually.

In contrast, the TmBiosciences fluorescent bead assay simultaneously andunambiguously characterized all the mutant sequences even when all weremixed together into one control tube (FIG. 6). At the same time, thenegative result for the R553X mutation when tested with the homozygousG551D control was 24% and the R553X mutation was 76%. All other negativenormal results were less than 16%. Obviously the G551D sequence wasdetected by the R553 normal sequence. This is due to the testmethodology used which relies on allele specific primer extension sincethe R553X mutation interferes with the G551 primer binding to minimizethe G551 signal but give a stronger signal on the normal R553 testplatform. Nevertheless, when the mechanism of this cross hybridizationis understood, the single artificial mixture of the 9 cloned fragmentscan be tested together and interpreted unambiguously as a singleTmBiosciences control reaction mixture (FIG. 6).

At the same time, these homozygous PCR amplified controls gaveheterozygous signals for mutation #3 and mutation #9 for the first 9homozygous mutations synthesized when tested on a multiplex cysticfibrosis tests from a third manufacturer. Although the manufacturercorrected the specificity of mutation #3 immediately upon learning ofour results, we moved on to the Innogenetics test system when we found asecond homozygous control with a heterozygous signal pattern among thefirst nine tested. This result emphasizes the importance of usinghomozygous controls over the heterozygous cell lines provided byCoriell. Obviously the third manufacturer had no way of knowing that ahomozygous sample could give a heterozygous result without anappropriate homozygous control. At the same time, when less than a fullpanel of mutation controls is tested on any one analytical run, smallchanges in hybridization conditions might modify hybridization of one ofthe 29 targets without changing the other 28 results. Our mixed set ofcontrols can detect both of these test specificity failures.

Discussion

Multiplex PCR amplification reactions typically cannot be relied upon toamplify each existing target site so that all can be visualized after atypical 10⁶-fold PCR amplification. For instance, one of us (RVL, unpub.results) developed a 15 site multiplex PCR test for Y chromosomedeletions that was assayed in 3 groups of 5 PCR target sites. Whencompleting 105 patient assays, 12 patients were identified and reportedwith deletions of one or more adjacent targets that were verified byrepeat individual PCR analysis at each site that failed to amplifyduring the initial screen. However, another 11 of the 105 samples hadindividual reactions that failed to amplify existing targets in two ormore sites that were physically separated by amplified sites. In thesesamples, the non-amplifying sites could always be amplified by repeatingthe initially failed PCR reaction with individual primer pairs to eachpreviously failed site with 0.1× or 10× of the total genomic DNAoriginally tested or by substituting repurified genomic DNA. Thus PCRamplifying all control targets together is critical in helping to assurethat multiplex PCR reactions are working effectively.

Our sequence verified controls are sufficiently long to serve asinternal simultaneous multiplex PCR amplification controls as well ashomozygous mutant sequence controls. Sequencing is considered the goldstandard of DNA sequence validation. Our cloned, sequenced controls arenested within sufficiently long CFTR gene fragments to include all thePCR primer sites save one site in eight commercial platforms (Aytay etal, Submitted). When using heterozygous cell lines, one has notconfirmed that the test system can distinguish unambiguously betweenhomozygous and heterozygous mutations. In contrast, Bajjani and Amoshave prepared multiple additional cystic fibrosis controls bysynthesizing and mixing 100 bp fragments. This approach does not controlfor multiplex PCR amplification of homozygous mutant sites, but doesrequire adding the control sequences to control tubes after PCRamplification, typically in a different laboratory location.Furthermore, these controls almost certainly consist of a mixture offragments with similar but not identical sequences. When synthesizing100 basepair control DNA fragments, the proportion of identicalsynthesized sequences typically drops precipitously as the synthesizedlength exceeds 60 basepairs. Thus the concentration of the syntheticprimers can only be estimated and the reliability of 100 bp synthesizedcontrols is anticipated to be less than perfect. Furthermore, when onerequires that the most robust controls are verified by sequencing,repeated bidirectional sequencing would need to be completed prior tousing any newly synthesized fragments to replace exhausted controlstocks. Since readable sequence typically begins about 30-40 basepairsdownstream from the end of the target, then 100 bp synthesized sequenceswould be verified in only 1 direction. In contrast, the entirebidirectional sequence can be determined for cloned controls by usingsequencing primers some distance into the vector from the cloning siteor into the adjacent cloned control fragment.

Clearly, homozygous controls for each mutant site are preferable to nocontrol and additional mutation controls prepared by this method can beused prior to developing cloned controls. However, each desiredsynthesized control can be readily prepared and cloned using fourselected primer sites and total normal genomic DNA that give a PCRproduct sufficient to control for PCR amplification. We have used thesecontrols since we first offered a 25 mutation multiplex cystic fibrosistest in our laboratory three years ago (Lebo et al. 2003). Preparing acontrol that works on multiple different commercial platforms requiredconsiderably more validation time prior to offering to the generalgenetics community (Aytay et al., 2005).

Multiple approaches can be used to synthesize heterozygous controls. Forinstance, normal genomic sequences can be PCR amplified directly fromtotal normal genomic template DNA using the same flanking primers likeF1 and R2 (FIG. 1, I-IV). These amplified normal sequences can then beverified by bidirectional sequence analysis through and beyond thevector's cloning site. Then normal cloned gene sequences can be mixedwith the cloned homozygous synthetic controls in equal copy number.Alternatively, Products #3 and #4 can be PCR amplified togethertypically in equal concentrations using forward primer F3 for theupstream mutation M3, reverse primer R1 for the M1/M2 mutations, and F1and R2 for the flanking primers (FIG. 1,XI-XIV; Lebo et al., unpublisheddata). Yet another approach would be to mix existing clones of normalgene regions with cloned synthesized homozygous controls.

In summary, we have synthesized 433 bp to 933 bp sequences containingthe 25 ACMG recommended mutation panel in 17 fragments inserted into 9vector cloning sites. The entire sequences and cloning sites have beenverified. These artificial mixtures can be added to test tubes adjacentto unknown samples in the PCR set up area to control for the multiplexPCR amplified cystic fibrosis test kits. Individual idiosyncrasies oftest formats for which these may be employed will depend upon theprecise PCR primer sites, whether these sites include normally variantsequences that interfere with PCR amplification, whether the primerpairs also amplify homologous genomic DNA sequences at other locations,and the relative amplification of each site during PCR at multipledifferent salt conditions found in multiple extracted DNA samples.Nevertheless, using this artificial mixture with all 25 mutations willsurely improve the reliability of each cystic fibrosis assay when run asa control with every assay of unknown patient samples.

This approach provides any laboratory with a ready means to producehomozygous and heterozygous controls for any laboratory assay withoutrequesting and sharing samples with other laboratories. When adding 32DNA tests to the menu of the Clinical Molecular Genetics Laboratory atBoston University, my laboratory typically spent 2-4 weeks to pick,order and test PCR primers and 2-4 months to obtain the requiredcontrols from generous colleagues. Synthesizing all unavailable controlDNA fragments and verifying the sequence provides a rapid means tosatisfy the most stringent laboratory reviewer and further expeditestest development for small groups of cooperating laboratories.Substantially larger numbers of multiplex controls can be offeredthrough facilities with the wherewithal to develop, produce, andmaintain the highest quality multiplex controls required to satisfy verylarge numbers of laboratories over wide geographical regions.

ELECTRONIC-DATABASE INFORMATION

-   Cystic Fibrosis Mutation Database, http://www    genet.sickkids.on.ca/cftr/-   Genetests, http://www.genetests.org/-   Online Mendelian inheritance in Man (OMIM), http://www.ncbi.nlm.nih    gov/OMIM/-   The Genome Database, http://www.gdb.org/

REFERENCES

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FIGURE LEGENDS

1. PCR Synthesis of Homozygous and Heterozygous Controls. Sections I-Xillustrates the incorporation of four defined homozygous mutations (M1,M2, M3, M4) into two different genomic fragments that are then splicedtogether for blunt end cloning. Sections XI-XV illustrate theincorporation of three defined heterozygous mutations (M1, M2, M3) intotwo different genomic fragments that when tested together give aheterozygous result. I. The first product to be synthesized has twomutations M1/M2 in a fragment with its upstream site defined by primerF1 and downstream site defined by primer R2 using total genomic TemplateDNA. II. PCR amplification incorporated mutations M1 and M2 intoupstream PCR amplified Product #1 using reverse primer R1 containing themutant sequences M1/M2 and forward upstream primer F1. III. In aseparate reaction mutations M1 and M2 were incorporated into downstreamPCR amplified Product #2 using forward primer F2 and reverse downstreamflanking primer R2. Then Products #1 and #2 are mixed with additional F2and R2 primers and amplified together to produce Product #3 (IV). V.Then mutation M3 was introduced into Product #3 using primers F1 and R3to amplify upstream Product #4 (VI). Similarly Primers F3 and R2 amplifydownstream Product #5 now with all three mutations M1, M2, and M3 (VII).Then Products #4 and #5 are spliced together with primers F1 and R2 tomake Product #6 (VII) with mutations M1, M2, M3 and upstream anddownstream sequences. Subsequently mutation M4 was introduced intoanother CFTR gene segment in an analogous fashion using M4mutation-specific primers along with flanking primers R2′F4 and R5 tomake Products #7 and #8 (not shown) and spliced product#9 (IX). ThenProducts #6 and #9 are spliced together by mixing these two productstogether and amplifying with F1 and R5 flanking primers to synthesizeProduct #10 (X) with mutations M1, M2, M3, and M4. These were blunt endcloned into a single vector (FIG. 2). XI. Alternatively, heterozygousproducts can be made using genomic template DNA, upstream and downstreamprimers (F1 and R2) and one primer for each site into which mutationswill be synthesized. XI-XII. Product #1A with a homozygous M1M2sequences is amplified with its own reverse primer R1 and forward primerF1. In a separate reaction homozygous Product #2A with mutation M3 isamplified using the forward M3 primer F3 and the reverse primer R2.These sites are selected so that PCR amplified Products #1A and #2A willhave overlapping homologous sequences. XV-XV. Then Products #2A and #3Aare mixed with forward primer F1 and reverse primer R2 and amplified togive Products #3A and #4A in the same reaction, typically in equalconcentrations. In fact, PCR amplifying with primers F1, F3, R1, and R2using total genomic template DNA also result in a mixture of Products#3A and #4A (Lebo et al. unpublished data).

2. Cloned homozygous CFTR gene controls. Seventeen fragments 433-933 bplong each with 1 to 4 CFTR mutations were prepared using PCR primerscontaining the mutant sites and flanking paired primers (See FIG. 1,I-VIII). Then 1 to 3 CFTR gene segments were joined (FIG. 1, IX-X) andblunt end cloned as a single fragment into 9 different vectors asillustrated: PCR amplification of the genomic DNA from one of theauthors also introduced 3 normal variant sites: 622-194G->A, 1525-61A-G,and 405+46G->T (CFTR Mutation Database). These 9 clones were preparedindependently so the desired combinations could be grown and themutation-carrying fragments mixed according to the selected commercialplatform requirements to which the control is applied.

3. Intron10/Exon11 Gene region with the sites of 5 synthesized mutationsillustrated. Because the G551D and R553X nucleotide substitution sitesare only separated by four basepairs, these mutations were added todifferent cloned fragments to avoid interference when tested in themultiple formats for which the synthesized mutations were intended.

4. Multiple Intronic Basepair Substitutions Downstream of A455E andN1303K in One Clone. The normal CFTR sequence is shown in the bottom(Subject) row and the initially analyzed cloned sequence in the top(Query) row. Top: The seven nucleotide substitutions found whensequencing the first exon9/intron 9 downstream clone were the same asthe substitutions found in the chromosome 20 CFTR pseudogene. All thenucleotide substitutions between the pseudogene and the active CFTR geneare indicated in bold above the top (Query) line. Bottom: The elevennucleotide substitutions found when sequencing the first exon 21/intron21 clone. These eleven substitutions occurred in a 130 bp repeatedsequence found 162 basepairs downstream from this site.

5. Innogenetics Nitrocellulose Filter Strip Results: Each of the cloneswas analyzed independently for the synthesized and cloned homozygousmutations. In every case the homozygous mutation was unambiguouslydistinguished (f1-f8). Note the positive test signal for the homozygous1717-1 mutation was more intense than the three homozygous signals forG542X, G551D, and R560T which are carried by the same PCR amplifiedfragment (f3,left). We interpret this signal intensity difference to bea characteristic of the Innogenetics multiplex PCR amplification, butthe results are readily interpretable. When these 9 clones are mixedtogether and tested, all the tested homozygous mutant sites gavehomozygous results (mix) except the G551D locus which is mutant in thetop cloned fragment (FIG. 1) and normal in the third cloned fragment(FIG. 1) with the R553X mutant site. Therefore the primer to the normalG551D site binds sufficiently to give a positive signal in the presenceof normal G551 sequence even though the R553X mutant sequence isupstream. However, the R553X primer only detects mutant sequence in thismixture and not the normal R553 signal on fragment 1 (FIG. 1) becausethe G551D signal interferes with the binding of the normal R553 reportermolecule in the test kit. In the end, two different Innogenetics teststrips define the specificity of all the homozygous controls at everysite except the G551D site which gives a heterozygous signal. In orderto have all multiplex test sites tested for the specificity of ahomozygous mutant result, fragments 1 and 3 (FIG. 1) must be preparedand tested in separate mixes of the other controls.

6. Entire Mixture of Homozygous Control Clones Tested on TmBiosciencesPlatform. Each homozygous control gives a clearly abnormal resultincluding the 5T locus which has 98% of the fluorescence among the 5T,7T, and 9T beads carrying specific oligonucleotides for each of theseloci (Left Panel, top 3 locations). At least 85% of the signal bound tothe homozygous location at all but one tested homozygous control site.Compare this to the 8 normal site results on these fragments where theTmBiosciences test assays for additional mutations (Left) and the normalDNA which gives only normal results at each tested site including thehomozygous 7T mutation.

1. A method of optimizing quality control in a genetic test assay, themethod comprising the steps of: testing for the presence of a normalgene nucleotide sequence portion at a pre-selected gene locus; testingfor the presence of at least a first mutant gene nucleotide sequenceportion at the pre-selected gene locus; and testing for interference byat least a first homologous nucleotide sequence portion.
 2. The methodof claim 1, wherein the genetic test assay is a hybridization basedassay.
 3. The method of claim 1, wherein the step of testing forinterference by homologous nucleotide sequence portion, involves thestep of providing a homologous nucleotide sequence control.
 4. Themethod of claim 3, wherein the homologous nucleotide sequence controlincludes at least a first homologous nucleotide sequence portion whichis homologous to the normal gene nucleotide sequence of the pre-selectedgene locus, and is devoid of a hybridizing nucleotide sequence portionof the pre-selected gene locus, wherein the hybridizing nucleotidesequence portion is sufficiently large to prevent detection of thepre-selected gene locus by the assay.
 5. The method of claim 4, whereinthe hybridizing nucleotide sequence portion is the normal genenucleotide sequence portion.
 6. The method of claim 4, wherein thehybridizing nucleotide sequence portion is substantially the entire genenucleotide sequence of the pre-selected gene locus.
 7. A controlcomprising: At least a first nucleotide sequence, wherein the at least afirst nucleotide sequence includes at least a first homologousnucleotide sequence portion and wherein the at least a first nucleotidesequence lacks a sufficiently large segment of the gene nucleotidesequence at an at least a first pre-selected gene locus to precludedetection of the gene nucleotide sequence at the at least a firstpre-selected gene locus by an assay, and Adapted for use in the assay,wherein the assay is for the detection of mutations in the genenucleotide sequence at the at least a first pre-selected gene locus. 8.The control of claim 7, wherein the at least a first nucleotide sequenceis total genomic DNA having the sufficiently large segment of the genenucleotide sequence at the at least a first pre-selected gene locusremoved.
 9. The control of claim 8 further comprising: at least a secondnucleotide sequence containing a sufficiently large segment of an atleast a first pre-selected mutant gene nucleotide sequence so as to bedetectable by the assay.
 10. The control of claim 9 wherein the at leasta second nucleotide sequence contains sufficiently large segments of atleast second and third pre-selected mutant gene nucleotide sequences soas to be detectable by the assay.
 11. The control of claim 10 whereinthe number of copies of the at least a first nucleotide sequence isapproximately equal to the number of copies of the at least a secondnucleotide sequence.
 12. A control comprising: a first nucleotidesequence portion, wherein the first nucleotide sequence portion includesat least a first homologous nucleotide sequence portion having asufficient length to adequately imitate corresponding normal or mutantnucleotide sequence portions found at a pre-selected gene locus beingtested in an assay and containing at least a first distinct nucleotidespecies, wherein the at least a first distinct nucleotide species is notfound in either the normal or mutant nucleotide sequence portions andwherein the at least a first distinct nucleotide species is suitablydistinct as to provide a means for confirmation that the firsthomologous nucleotide sequence portion is not being detected in theassay by a normal or a mutant sequence primer used in the assay; and asecond nucleotide sequence portion, wherein the second nucleotidesequence portion includes a sufficiently large segment of a first mutantgene nucleotide sequence portion found at the pre-selected gene locus soas to be detectable by the assay.
 13. The control of claim 12, furthercomprising: at least a second homologous nucleotide sequence portionsubstantially adjacent the first nucleotide sequence portion, whereinthe at least a second homologous nucleotide sequence portion has asufficient length to adequately imitate corresponding normal or mutantnucleotide sequence portions of the pre-selected gene locus being testedin the assay and containing at least a first distinct nucleotidespecies, wherein the at least a first distinct nucleotide species is notfound in either the normal or mutant nucleotide sequence portions andwherein the at least a first distinct nucleotide species is suitablydistinct as to provide a means for confirmation that the at least asecond homologous nucleotide sequence portion is not being detected inthe assay by a normal or a mutant sequence primer used in the assay 14.The control of claim 13, wherein the second nucleotide sequence portionfurther comprises: A sufficiently large segment of an at least a secondmutant gene nucleotide sequence portion found at the pre-selected genelocus so as to be detectable by the assay